Injury to podocytes leads to the onset of chronic renal diseases characterized by proteinuria. Elevated transforming growth factor (TGF)-β in kidney tissue is associated with podocyte damage that ultimately results in apoptosis and detachment. We investigated the proapoptotic mechanism of TGF-β in immortalized mouse podocytes. Exogenous TGF-β1-induced podocyte apoptosis through caspase-3 activation, which was related to elevated ROS levels generated by selective upregulation of NADPH oxidase 4 (Nox4). In mouse podocytes, Nox4 was predominantly localized to mitochondria, and Nox4 upregulation by TGF-β1 markedly depolarized mitochondrial membrane potential. TGF-β1-induced ROS production and caspase activation were mitigated by an antioxidant, the Nox inhibitor diphenyleneiodonium, or small interfering RNA for Nox4. A TGF-β receptor I blocker, SB-431542, completely reversed the changes triggered by TGF-β1. Knockdown of either Smad2 or Smad3 prevented the increase of Nox4 expression, ROS generation, loss of mitochondrial membrane potential, and caspase-3 activation by TGF-β1. These results suggest that TGF-β1-induced mitochondrial Nox4 upregulation via the TGF-β receptor-Smad2/3 pathway is responsible for ROS production, mitochondrial dysfunction, and apoptosis, which may at least in part contribute to the development and progression of proteinuric glomerular diseases such as diabetic nephropathy.

A low level of circulating irisin is a sensitive marker for muscle weakness and atrophy. Irisin is a potential biomarker for muscle dysfunction that could help predict the onset of sarcopenia and provide new avenues for monitoring age-related muscle changes. Geriatr Gerontol Int 2017; 17: 2266-2273.

Inorganic phosphate (Pi) plays an important role in cell signaling and energy metabolism. In insulin-releasing cells, Pi transport into mitochondria is essential for the generation of ATP, a signaling factor in metabolism-secretion coupling. Elevated Pi concentrations, however, can have toxic effects in various cell types. The underlying molecular mechanisms are poorly understood. Here, we have investigated the effect of Pi on secretory function and apoptosis in INS-1E clonal β-cells and rat pancreatic islets. Elevated extracellular Pi (1~5 mM) increased the mitochondrial membrane potential (ΔΨm), superoxide generation, caspase activation, and cell death. Depolarization of the ΔΨm abolished Pi-induced superoxide generation. Butylmalonate, a nonselective blocker of mitochondrial phosphate transporters, prevented ΔΨm hyperpolarization, superoxide generation, and cytotoxicity caused by Pi. High Pi also promoted the opening of the mitochondrial permeability transition (PT) pore, leading to apoptosis, which was also prevented by butylmalonate. The mitochondrial antioxidants mitoTEMPO or MnTBAP prevented Pi-triggered PT pore opening and cytotoxicity. Elevated extracellular Pi diminished ATP synthesis, cytosolic Ca(2+) oscillations, and insulin content and secretion in INS-1E cells as well as in dispersed islet cells. These parameters were restored following preincubation with mitochondrial antioxidants. This treatment also prevented high-Pi-induced phosphorylation of ER stress proteins. We propose that elevated extracellular Pi causes mitochondrial oxidative stress linked to mitochondrial hyperpolarization. Such stress results in reduced insulin content and defective insulin secretion and cytotoxicity. Our data explain the decreased insulin content and secretion observed under hyperphosphatemic states.

Background: Mitochondrial Ca 2ϩ uptake affects energy metabolism and insulin secretion. Results: Knockdown of mitochondrial Ca 2ϩ uniporter decreases respiratory chain activity and mitochondrial pH gradient generation. Conclusion: Mitochondrial Ca 2ϩ uptake via uniporter is essential for oxidative phosphorylation and metabolism-secretion coupling. Significance: The present study identifies mechanisms of action and bioenergetic consequences of mitochondrial Ca 2ϩ transporters in insulin-releasing cells.

Hyperphosphatemia is the primary risk factor for vascular calcification, which is closely associated with cardiovascular morbidity and mortality. Recent evidence showed that oxidative stress by high inorganic phosphate (Pi) mediates calcific changes in vascular smooth muscle cells (VSMCs). However, intracellular signalings responsible for Pi-induced oxidative stress remain unclear. Here, we investigated molecular mechanisms of Pi-induced oxidative stress related with intracellular Ca2+ ([Ca2+]i) disturbance, which is critical for calcification of VSMCs. VSMCs isolated from rat thoracic aorta or A7r5 cells were incubated with high Pi-containing medium. Extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin were activated by high Pi that was required for vascular calcification. High Pi upregulated expressions of type III sodium-phosphate cotransporters, PiT-1 and -2, and stimulated their trafficking to the plasma membrane. Interestingly, high Pi increased [Ca2+]i exclusively dependent on extracellular Na+ and Ca2+ as well as PiT-1/2 abundance. Furthermore, high Pi induced plasma membrane depolarization mediated by PiT-1/2. Pretreatment with verapamil, as a voltage-gated Ca2+ channel (VGCC) blocker, inhibited Pi-induced [Ca2+]i elevation, oxidative stress, ERK activation and osteogenic differentiation. These protective effects were reiterated by extracellular Ca2+ free condition, intracellular Ca2+ chelation or suppression of oxidative stress. Mitochondrial superoxide scavenger also effectively abrogated ERK activation and osteogenic differentiation of VSMCs by high Pi. Taken together, we suggest that high Pi activates depolarization-triggered Ca2+ influx via VGCC, and subsequent [Ca2+]i increase elicits oxidative stress and osteogenic differentiation. PiT-1/2 mediates Pi-induced [Ca2+]i overload and oxidative stress, but in turn, PiT-1/2 is upregulated by consequences of these alterations.